The Ancestor and Evolution of the Giant Muscle Protein Connectin/Titin

The emergence of connectin, also called titin, a muscular spring and the largest protein in living organisms, is critical in metazoan evolution as it enables striated muscle-based locomotion. However, its evolutionary history remains unclear. Here, we investigated the evolutionary process using genomic analysis and deduced the ancestor of connectin. The region between the HOX and WNT clusters in the human genome, where the connectin gene (CON (TTN)) is located, was quadrupled by two rounds of whole-genome duplication (WGD) in the ancestor of jawed vertebrates. However, connectin ohnologs were deleted before the advent of jawed vertebrates, resulting in a single connectin gene. Additionally, one of the connectin ohnologs created by the third round of teleost WGD disappeared, while the other was duplicated on the same chromosome. We also discovered that the connectin and connectin family genes consistently underwent local duplication on the same chromosome, though the underlying mechanism remains unknown. Using synteny analysis, we identified KALRN and its ohnolog TRIO as putative ancestral paralogs of the connectin gene. TRIO/KALRN has a connected structure of SESTD1-CCDC141-CON (TTN), and its synteny is conserved in the Drosophila genome. Furthermore, we confirmed that this connected structure, termed 'connectitin,' (connected-connectin/titin) is conserved in cnidarians and placozoans. Molecular phylogenetic analysis revealed that it diverged from TRIO/KALRN prior to the emergence of these animals, suggesting that metazoan muscle may have a single origin. These findings enhance our understanding of the evolutionary processes of striated muscles in the animal kingdom.